Project Summary/Abstract Micro-RNAs (miRNA) are an abundant class of 22-nucleotide regulatory elements that guide the silencing of mammalian genes and contribute to broad range of cellular processes including developmental timing, life span, brain development, cell proliferation, and cancer. A critical step in the miRNA pathway is the assembly of miRNAs into an active form, a large ribonucleoprotein complex called RISC. This project aims to characterize the assembly of RISC with an emphasis on understanding the structural details underlying processing and loading of miRNAs into RISC. Research is focused on a three-protein assembly termed the RISC-loading complex (RLC). Using a combination of biochemistry and structural biology we will identify key features of the RLC mechanism that may be exploited for therapeutic purposes. Biochemical efforts will focus on dissecting the mechanisms underlying miRNA recognition and processing efficiency. Electron microscopy will be used to determine the molecular architecture of the RLC, which will guide further biochemical experiments. We aim to determine molecular structures of the RLC stalled at key steps in RISC-loading to fully visualize this essential cellular process. These combined structural and functional studies will provide comprehensive mechanistic insights into one of the most fundamental but least understood pathways of controlling mammalian gene expression. Project Narrative RNA interference (RNAi) is a natural process by which human genes are turned off, or silenced, which is involved in biological functions ranging from life span, to brain development, to cancer. This project aims to understand the molecular machinery that decides which genes will be silenced by RNAi. Results will contribute to on-going efforts to harness the RNAi process as a powerful new type of therapeutic for the treatment of human disease.